Sepsis is morbid condition induced by a toxin, the introduction or accumulation of which is most commonly caused by infection or trauma. The initial symptoms of sepsis typically include chills, profuse sweat, irregularly remittent fever, prostration and the like, followed by persistent fever, hypotension leading to shock, neutropenia, leukopenia, disseminated intravascular coagulation, adult respiratory distress syndrome and multiple organ failure.
Sepsis-inducing toxins have been found associated with pathogenic bacteria, viruses, plants and venoms. Among the well described bacterial toxins are the endotoxins or lipopolysaccharides(LPS) of the gram-negative bacteria. These molecules are glycolipids that are ubiquitous in the outer membrane of all gram-negative bacteria. While the chemical structure of most of the LPS molecule is complex and diverse, a common feature is the lipid A region of LPS [E. Th. Rietschel et al., in Handbook of Endotoxins, 1:187-214 eds., R. A. Proctor and E. Th. Rietschel, Elsevier, Amsterdam (1984)]; recognition of lipid A in biologic systems initiates many, if not all, of the pathophysiologic changes of sepsis. Because lipid A structure is highly conserved among all types of gram-negative organisms, common pathophysiologic changes characterize gram-negative sepsis.
LPS is believed to be a primary cause of death in humans during gram-negative sepsis, particularly when the symptoms include adult respiratory distress syndrome (ARDS) [van Deventer et al., Lancet, 1:605 (1988); ziegler et al., J. Infect. Dis., 136:19-28 (1987)]. For instance, one particular cytokine, tumor necrosis factor alpha/cachectin (TNF), has recently been reported to be a primary mediator of septic shock [Beutler et al., N. Eng. J. Med., 316:379 (1987)]. Intravenous injection of LPS endotoxin from bacteria into experimental animals and man produces a rapid, transient release of TNF [Beutler et al., J. Immunol., 135:3972 (1985); Mathison et al., J. Clin. Invest. 81:1925 (1988)]. Evidence that TNF is a critical mediator of septic shock comes primarily from experiments in which pretreatment of animals with anti-TNF antibodies reduces lethality [Beutler et al., Science, 229:869, (1985); Mathison et al., J. Clin. Invest, 81:1925 (1988)]. These reports suggest that interruption of the secretion of TNF caused by LPS or other factors would ameliorate the often lethal symptoms of sepsis.
Current concepts support the contention that the primary response of the host to LPS (including man) involves the recognition of LPS by cells of the monocyte/macrophage lineage, followed by the rapid elaboration of a variety of cell products including the general group known as cytokines. Other cell types believed to participate in sepsis and in particular in the response to LPS are polymorphonuclear leukocytes (PMN) and endothelial cells; each of these cell types are also capable of responding to LPS with the elaboration of potent inflammatory substances, and in the case of polymorphonuclear leukocytes, the elaboration of cytotoxic molecules.
Upon introduction of LPS into the blood, it may bind to a protein termed lipopolysaccharide binding protein (LBP). LBP is a 60 kD glycoprotein present at concentrations of less than 100 ng/ml in the serum of healthy animals and man. During the acute phase, LBP is synthesized by hepatocytes, and reaches concentrations of 30-50 ug/ml in serum. LBP can be purified from acute phase human and rabbit serum [Tobias et al. J. Exp. Med., 164:777-793 (1986)]. LBP recognizes the lipid A region of LPS and forms high affinity, 1:1 stoichiometric complexes with both rough and smooth form LPS [Tobias et al., J. Biol. Chem., 264:10867-10871 (1989)]. LBP bears N-terminal sequence hemology with the LPS-binding protein known as bactericidal permeability-increasing factor, (BPI) [Tobias et al., J. Biol. Chem., 263:13479-13481, (1988)]. BPI is stored in the specific granules of PMN [Weiss et al., Blood, 69:652-659, (1987)] and kills gram-negative bacteria by binding LPS and disrupting the permeability barrier [Weiss et al., J. Immunol., 32:3109-3115, (1984)]. In contrast to BPI, LBP is not directly cytotoxic for gram-negative bacteria [Tobias et al., J. Biol. Chem., 263:13479-13481, (1988)] and its precise biological function has been obscure.
By way of further background, the cells of the monocyte/macrophage lineage perform diverse immune function including the phagocytosis of micro-organisms, the uptake of antigenic material and its presentation in a form which is stimulatory to helper T cells. They are probably also involved in the immune surveillance against tumors and they secrete some complement components and cytokines. Surface membrane antigens play a critical role in regulating these activities. Several monocyte/macrophage surface antigens have been identified and their molecular weight has been determined. One such antigen, CD14, is a 55-kD glycoprotein expressed by monocytes, macrophages, and activated granulocytes. It is recognized by a number of monoclonal antibodies (mAbs) including MO2, MY4, 3C10 and LEUM3. Although no biological function has yet been ascribed to CD14, its restricted expression on mature cells suggests an important effector function. The nucleotide sequence of the gene encoding the monocyte cell surface differentiation antigen CD14 has been determined and the amino acid residue sequence of CD14 has been deduced therefrom [Ferrero et al., Nucleic Acids Research, 16:4173 (1988)].
Human serum contains trace quantities of the protein lipopolysaccharide binding protein (LBP), and it has recently been shown that this protein interacts first with LPS (endotoxin) and then with CD14 on the surface of phagocytes to provoke cellular responses that underlie the phenomenon of septic shock [Wright et al., Science, 249:1431-1433 (1990)]. However, several observations suggested that, while LBP can participate in the binding of LPS and CD14, it may not be the only protein involved. Three observations have been noted that commend this conclusion: (1) the addition of purified LBP to human mononuclear cells did not restore the ability of these cells to synthesize TNF in response to physiological doses of LPS as observed in the presence of serum; (2) LBP is an acute phase reactant that is present in satisfactory quantities during the acute phase response, but sufficient quantities may not be present in healthy individuals to explain their responses to LPS; and (3) "LPB-like" activity has been assayed by measuring the ability of solutions to mediate the binding of LPS to macrophages, and very high levels in serum from healthy individuals have been found, which levels are far too high to be explained by the content of LBP in the serum. These observations were the incentive for seeking a novel molecule in human serum that had-the properties of binding LPS and CD14.
In copending U.S. application Ser. No. 07/473,609, now abandoned, a molecule named "septin" by the Applicant was identified that was found to function as an opsonin, and that exhibited the following profile of characteristics:
(a) the opsonin is capable of binding to lipopolysaccharide (LPS) to form a complex that is recognized by a receptor or monocytes, macrophage cells and polymorphonuclear cells (PMNs); PA1 (b) the opsonin possess an apparent molecular weight of about 90 kD as determined by SDS-PAGE; PA1 (c) the opsonin is present in high levels in normal serum, and enhances the elaboration of TNF by monocytes in response to LPS; PA1 (d) a complex of the opsonin and LPS stimulates PMN activity, promotes increased PMN adhesion to endothelium, causes PMN degranulation and upregulates the expression of the adhesion molecule CR3; and PA1 (e) the opsonin is unable to bind to the receptor CD14 when first bound in a complex with Lipid IVa, the biosynthetic precursor of LPS.
The apparent involvement of the molecule with host response to invasion and other traumatic events forecast an active role for the molecule in both diagnostic and therapeutic applications related to infection and trauma. Subsequent study of the molecule has revealed additional structural and functional characteristics that enhance the understanding of the molecule and, in turn, are believed to broaden its utility. It is therefore toward the increased understanding and application of the molecule that the present Application is directed.